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Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, more nucleotides will increase the difficulties in plasmid construction and the risk of wrong integration in genome such as base-pair or fragment missing (Gheysen et al., 1990). And also, in many organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly (Sun et al., 2015). Thus, we have developed a method using single transcriptional unit (STU) CRISPR-Cas9 system to drive the expression of both Cas9 and sgRNAs from a single RNA polymerase II promoter to achieve effective genome editing in plants.

The sgRNA of the CRISPR-Cas9 system is mainly promoted by the small nuclear RNA promoters such as U6 and U3. Although it has been tested with prospered efficiency in many cases, it also has some limitations: (1) it is hard to achieve coordinated and/or inducible expression of Cas9 and the sgRNAs; (2) manipulating multiple sgRNAs for multiplexed gene editing can be tedious, requiring multiple Pol III promoters. The traditional RNA polymerase II promoter can’t be used in driving sgRNA expression, extra nucleotides will be added to the 5’- and 3’-ends of gRNA by RNA polymerase II and may interrupt the normal gRNA function. Additionally, RNAs transcribed by RNA polymerase II are exported rapidly into the cytoplasm while nuclear localization is required for the CRISPR-Cas9/gRNA duplex to access the genome editing (Lei et al., 2001). To overcome these obstacles, we use the ribozyme’s self-catalyzed cleavage to release the precise processing mature sgRNA under a RNA polymerase II promoter which drives expression of both Cas9 and sgRNA (named STU CRISPR-Cas9 system, Figure 1). Compared to the traditional small nuclear RNA promoters used in sgRNA expression, our STU CRISPR-Cas9 system has some advantages: (1) it’s shorter and easier in vector construction, and it will increase the transformation efficiency under some circumstances; (2) it only needs extra ribozyme flanking sequence (shorter than any RNA polymerase III promoter we are currently using) for multiple sgRNAs expression;(3) it has shown higher deletion efficiency induced by double sgRNAs. Thus, the STU CRISPR-Cas9 system driven by a single RNA polymerase II promoter can replace the traditional CRISPR-Cas9 system now we are using whether in vivo or in vitro if appropriate promoters are chosen.

Figure 1. Schematic illustration of the single transcription unit (STU) CRISPR-Cas9 system. Once transcribed by a Pol II promoter, the STU CRISPR-Cas9 primary transcripts will undergo self-cleavage by hammerhead ribozyme (RZ) to release the mature Cas9 mRNA and sgRNA. The Cas9 mRNA is terminated with a synthetic polyA (pA) sequence to facilitate translation, The RZ sequence (in blue) and its target sequence (in black) are illustrated.

Select appropriate sgRNA targets for the genes of interest using the online sgRNA design tools such as CRIPSR-P v2.0 (http://cbi.hzau.edu.cn/CRISPR2/); CRISPR RGEN tools (http://www.rgenome.net/cas-offinder/); E-CRISP (http://www.e-crisp.org/E-CRISP/designcrispr.html). These are several web-based tools available for sgRNA design. They have mainly the same functions in sgRNA design and Off-target prediction. The main difference is the algorithm of the scoring system.Notes: sgRNA targets containing a restriction enzyme site at the Cas9 cleavage site would contribute to identify mutant using polymerase chain reaction-restriction endonuclease digestion assay.

Design and order forward and reverse oligonucleotides for cloning sgRNA into the STU CRISPR-Cas9 expression vector. (1) the forward sgRNA oligonucleotide contains a ‘CGGA’ sequence at the 5’ end followed by 20 bases of sgRNA targets without PAM sites (N20); (2) the reverse sgRNA oligonucleotide contains an ‘AAAC’ at the 5’ end followed by the reverse complement of N20.
For example, if the target site is GTTGGTCTTTGCTCCTGCAGAGG (AGG is PAM), the forward and reverse oligonucleotides should be:
Forward oligonucleotide: 5’-CGGAGTTGGTCTTTGCTCCTGCAG-3’
Reverse oligonucleotide: 5’-AAACCTGCAGGAGCAAAGACCAAC-3’

Annealing of sgRNA oligos

Mix 10 µl of the forward and reverse oligos (100 µM) of each sgRNA in separate microtubes.

Incubate the microtubes at 95 °C for 5 min in a heating block or thermal cycler.

Allow the microtubes to slowly cool down to room temperature.

Make a 1:200 dilution of the annealed mixture with deionized water.

Vector cloning
Two methods could be used to clone sgRNAs into the STU CRISPR-Cas9 expression vector: Cut and ligation or Golden Gate method (Figure 2). These two methods are the same in procedure including BsaI digestion and T4 DNA ligase ligation. The Golden Gate reaction is much easier because the digestion and ligation will process in one PCR tube, and it may save some time.

Figure 2. Schematic illustration of the cloning procedure described in the protocol

Verify the positive clones by colony PCR and Sanger sequencing. Colony PCR can be performed with the forward sgRNA oligonucleotide (e.g., see step A2) and ZY065-RB: (5’-ttctaataaacgctcttttctct-3’). The expected product size is approximately 230 bp. ZY065-RB can be used for sequencing.

Two sgRNAs can be cloned into the STU CRISPR-Cas9 expression vector to target two sites simultaneously (Figure 2).

Design two primers as follows:BsaI-sgRNA01-F: 5’-CAGGTCTCACGGA-N20-gttttagagctagaaatagcaagttaa-3’BsaI-sgRNA02-R: 5’-TCGGTCTCCAAAC-N20-tccggtgacaaaagcaccga-3’GGTCTC is the BsaI recognition sequence;
‘N20’ is same as the sgRNA01 target-specific sequence;
‘N20’ is the reverse complement of the sgRNA02 target-specific sequence;
The lowercase letters are complementary with the STU CRISPR-Cas9 expression vector.Note: PAGE purified oligos are highly recommended, desalted is also OK.

Set up a 50 μl PCR reaction to amplify DNA for STU CRISPR-Cas9 expression vector construction.

Our STU CRISPR-Cas9 system has the potential for multiplex sites genome editing. For more than two sites within one STU CRISPR-Cas9 vector, two-round PCR could be performed to clone different sgRNAs into the expression vector (Figure 2).

Examples of STU CRISPR-Cas9 system application including gene editing and gene deletion with sequencing data in rice, tobacco and Arabidopsis can be found in the original paper (Tang et al., 2016; Link to paper). Additionally, diagrams of the procedure, as well as examples of genome editing and sgRNA multiplex construction, can also be found in the original research paper (Tang et al., 2016).

Notes

If failed to get colony using Golden Gate method, increase the number of cycles to 15-20 times.

We tested the STU CRISPR-Cas9 system in several organisms (including rice, tobacco and Arabidopsis) and successfully achieved efficient genome editing.

Y.Z. was supported by grants from the National Science Foundation of China (31330017 and 31371682), the Sichuan Youth Science and Technology Foundation (2017JQ0005) and the Fundamental Research Funds for the Central Universities (ZYGX2016J119 and ZYGX2016J122). This protocol is developed based on our previous study published in Molecular Plant (Tang et al., 2016).

How to cite: Tang, X., Zhong, Z., Zheng, X. and Zhang, Y. (2017). Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants. Bio-protocol 7(17): e2546. DOI: 10.21769/BioProtoc.2546.

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